Mesenchymal stem cell-derived extracellular vesicles and their use

Abstract

This invention relates to extracellular vesicles derived from human mesenchymal stem cells and their use. The extracellular vesicles derived from mesenchymal stem cells according to the present invention inhibit the expression of fibrosis-related proteins, wound closure, and cell infiltration, which are increased in lung epithelial cells due to TGF-β1 treatment. In addition, microRNAs contained in large quantities in the extracellular vesicles inhibit the gene expression of fibrosis-related proteins in lung epithelial cells and lung fibroblasts. These extracellular vesicles significantly reduce the expression of fibrosis-related proteins, collagen production, and fibrotic areas in a mouse model of pulmonary fibrosis. Therefore, the extracellular vesicles according to the present invention can be used to prevent and treat pulmonary fibrosis.

Description

[Technical Field]

[0001] This invention relates to extracellular vesicles derived from human mesenchymal stem cells and their use. [Background technology]

[0002] Idiopathic pulmonary fibrosis (IPF) is the most common form of idiopathic interstitial pneumonia (IIP), a type of interstitial lung disease (ILD), although its exact cause remains unknown. Chronic pro-inflammatory fibrosis was once thought to be the primary cause of Idiopathic pulmonary fibrosis.

[0003] Pirfenidone and nintedanib, approved by the FDA in 2014, are used as treatments for idiopathic pulmonary fibrosis. However, these treatments do not address the underlying cause and have been reported to cause tolerability issues and side effects. In addition, drugs currently under development through clinical trials are monophasic, raising concerns about their efficacy and side effects. Therefore, there is a need for new treatments with multiple mechanisms of action.

[0004] Mesenchymal stem cells have been reported to be effective in treating idiopathic pulmonary fibrosis through their immunomodulatory, anti-inflammatory, and epithelial cell regeneration-promoting effects (Yang et al., Front Cell Dev Biol. (2021) 9:639657; Xie et al., Front Pharmacol. (2021) 11:590972). However, the biggest challenge with stem cells is that engraftment and survival rates vary among patients, making it difficult to accurately demonstrate their effectiveness. Furthermore, stem cells may transform into cancer cells. Therefore, research on treatments for pulmonary fibrosis utilizing mesenchymal stem cells is needed. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Therefore, the inventors conducted research to develop a therapeutic agent for pulmonary fibrosis. As a result, the inventors found that extracellular vesicles isolated from mesenchymal stem cell culture medium showed excellent efficacy in treating pulmonary fibrosis in a pulmonary fibrosis model mouse, thereby completing the present invention. [Means for solving the problem]

[0006] To achieve the above objective, in one aspect of the present invention, an extracellular vesicle is provided containing one overexpressed microRNA selected from the group consisting of hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-let-7f-5p, and combinations thereof.

[0007] In another embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis is provided, comprising extracellular vesicles as an active ingredient.

[0008] In another embodiment of the present invention, the use of extracellular vesicles for the prevention or treatment of fibrosis is provided.

[0009] In another aspect of the present invention, a method for preventing or treating fibrosis is provided, comprising the step of administering an agent targeting extracellular vesicles. [Effects of the Invention]

[0010] The extracellular vesicles derived from mesenchymal stem cells according to the present invention inhibited the expression of fibrosis-related proteins, wound closure, and cell invasion, which were increased by TGF-β1 treatment in lung epithelial cells. In addition, microRNAs contained in large quantities in the extracellular vesicles inhibited the gene expression of fibrosis-related proteins in lung epithelial cells and lung fibroblasts. These extracellular vesicles significantly reduced the expression of fibrosis-related proteins, collagen production, and fibrotic areas in a pulmonary fibrosis model mouse. Therefore, the extracellular vesicles according to the present invention can be used to prevent and treat pulmonary fibrosis. [Brief explanation of the drawing]

[0011] [Figure 1] This is a schematic diagram illustrating extracellular vesicles secreted from human mesenchymal stem cells (hMSCs) derived from various human tissues and the use of extracellular vesicles in the treatment of pulmonary fibrosis. [Figure 2] This is a schematic diagram illustrating a method for isolating extracellular vesicles derived from human mesenchymal stem cells according to one embodiment of the present invention. [Figure 3] This graph and table show the particle size distribution and concentration of extracellular vesicles (hMSC-EVs) isolated from the culture medium of human umbilical cord-derived mesenchymal stem cells, as measured using a nanoparticle tracking analyzer according to one embodiment of the present invention. [Figure 4] This figure shows the results obtained by observing the bilipid membrane structure of hMSC-EV according to one embodiment of the present invention using a cryogenic transmission electron microscope. [Figure 5] This figure shows the results obtained by identifying markers in hMSC-EV using Western blotting according to one embodiment of the present invention. [Figure 6] This graph shows the results obtained by identifying the phenotype of hMSC-EV using flow cytometry according to one embodiment of the present invention. [Figure 7a] Figures 7a and 7b are graphs showing the results obtained by analyzing the intrinsic factors of hMSC-EV using small molecule RNA sequencing according to one embodiment of the present invention. [Figure 7b] Figures 7a and 7b are graphs showing the results obtained by analyzing the intrinsic factors of hMSC-EV using small molecule RNA sequencing according to one embodiment of the present invention. [Figure 8] This graph shows the results of q-PCR identification of fibrosis-related gene expression after treating human lung epithelial cells (A549 cells) with TGF-β1 to induce EMT (epithelial-mesenchymal transition), and then treating the A549 cells with hMSC-EV. [Figure 9]This is a figure showing the results obtained by treating human lung epithelial cells (A549 cells) with TGF-β1 to induce EMT, and then identifying the wound healing effect after pretreatment of A549 cells with hMSC-EV. [Figure 10] This is a figure and graph showing the results obtained by treating human lung epithelial cells (A549 cells) with TGF-β1 to induce EMT, and then identifying the degree of cell migration and cell invasion after pretreatment of A549 cells with hMSC-EV. [Figure 11] This is a figure showing the results obtained by treating human lung epithelial cells (A549 cells) with TGF-β1 to induce EMT, and then identifying the expression of fibrosis-related proteins after pretreatment of A549 cells with hMSC-EV using Western blotting. [Figure 12] This is a figure showing the results obtained by treating human lung epithelial cells (A549 cells) with TGF-β1 to induce EMT, and then identifying the expression of fibronectin after pretreatment of A549 cells with hMSC-EV using cell immunofluorescence staining. [Figure 13] This is a graph showing the results obtained by identifying the expression of microRNAs after overexpression of miR-148a-3p, miR-143-3p, miR-100-5p, Let-7a-5p, Let-7b-5p, or Let-7f-5p in human lung epithelial cells (A549 cells) using q-PCR. [Figure 14] This is a graph showing the results obtained by identifying the expression of microRNAs after overexpression of miR-148a-3p, miR-143-3p, miR-100-5p, Let-7a-5p, Let-7b-5p, or Let-7f-5p in human lung fibroblasts (NHLF cells) using q-PCR. [Figure 15a]Figures 15a and 15b are graphs showing the results obtained by identifying the gene expression of fibrosis-related factors using q-PCR after overexpression of miR-148a-3p, miR-143-3p, miR-100-5p, Let-7a-5p, Let-7b-5p, or Let-7f-5p in human lung epithelial cells (A549 cells) followed by treatment with TGF-β1. [Figure 15b] Figures 15a and 15b are graphs showing the results obtained by identifying the gene expression of fibrosis-related factors using q-PCR after overexpression of miR-148a-3p, miR-143-3p, miR-100-5p, Let-7a-5p, Let-7b-5p, or Let-7f-5p in human lung epithelial cells (A549 cells) followed by treatment with TGF-β1. [Figure 16] This graph shows the results obtained by identifying the gene expression of fibrosis-related factors using q-PCR after overexpression of miR-148a-3p, miR-143-3p, miR-100-5p, Let-7a-5p, Let-7b-5p, or Let-7f-5p in human lung fibroblasts (NHLF cells) and treatment with TGF-β1. [Figure 17] This figure shows the results obtained by identifying the expression of fibrosis-related proteins in human lung epithelial cells (A549 cells) after overexpression of miR-148a-3p, miR-143-3p, miR-100-5p, Let-7a-5p, Let-7b-5p, or Let-7f-5p, following treatment with TGF-β1, using Western blotting. [Figure 18] This figure shows the results obtained by identifying the expression of fibrosis-related proteins in human lung fibroblasts (NHLF cells) after overexpression of miR-148a-3p, miR-143-3p, miR-100-5p, Let-7a-5p, Let-7b-5p, or Let-7f-5p, following treatment with TGF-β1, using Western blotting. [Figure 19]This is a schematic diagram showing the experimental schedule for identifying the therapeutic effect of hMSC-EV on pulmonary fibrosis in a bleomycin-induced pulmonary fibrosis animal model (BLM-induced C57BL / 6 mouse). [Figure 20] This graph shows the results of q-PCR analysis of gene expression of fibrosis-related factors in mouse lung tissue 8 days after a single dose of hMSC-EV in an animal model of bleomycin (BLM)-induced pulmonary fibrosis. [Figure 21] This graph shows the results obtained by measuring the concentration of soluble collagen in mouse lung tissue 8 days after a single dose of hMSC-EV in an animal model of bleomycin (BLM)-induced pulmonary fibrosis. [Figure 22] This figure shows the results obtained by observing lung tissue using Masson's trichrome staining 8 days after a single dose of hMSC-EV in an animal model of bleomycin (BLM)-induced pulmonary fibrosis. Magnification: 200x, Scale bar: 50 μm. [Figure 23] This figure shows an experimental schedule for identifying the therapeutic effect of hMSC-EV on pulmonary fibrosis in an animal model of bleomycin (BLM)-induced pulmonary fibrosis. [Figure 24] This figure shows the results obtained by observing the morphology of mouse lungs extracted after administration of hMSC-EV in an animal model of bleomycin (BLM)-induced pulmonary fibrosis. [Figure 25] This graph shows the results obtained by identifying the gene expression of fibrosis-related factors in mouse lung tissue after administration of hMSC-EV in a bleomycin (BLM)-induced pulmonary fibrosis animal model using q-PCR. [Figure 26a] This graph shows the results obtained by identifying the expression of fibrosis-related proteins in mouse lung tissue after administration of hMSC-EV in a bleomycin (BLM)-induced pulmonary fibrosis animal model using Western blotting. [Figure 26b]This figure shows the results obtained by identifying the expression of fibrosis-related proteins in mouse lung tissue after administration of hMSC-EV in a bleomycin (BLM)-induced pulmonary fibrosis animal model using Western blotting. [Figure 27] This graph shows the results obtained by identifying the concentration of soluble collagen in mouse lung tissue after administration of hMSC-EV in an animal model of bleomycin (BLM)-induced pulmonary fibrosis. [Figure 28] These figures and graphs show the results of hematoxylin and eosin staining (Figure) and Ashcroft score analysis (Graph) of mouse lung tissue after administration of hMSC-EV in an animal model of bleomycin (BLM)-induced pulmonary fibrosis. [Figure 29] This figure and graph show the results obtained by identifying collagen regions in mouse lung tissue after administration of hMSC-EV in a bleomycin (BLM)-induced pulmonary fibrosis animal model using Masson's trichrome staining. X40: 40x magnification, X200: 200x magnification. [Modes for carrying out the invention]

[0012] Extracellular vesicles In one aspect of the present invention, an extracellular vesicle is provided containing one overexpressed microRNA selected from the group consisting of hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-let-7f-5p, and combinations thereof. In this case, the cell may be a human-derived cell, such as a human tissue, cell line, or stem cell, and more specifically, may be a stem cell.

[0013] Cells derived from human tissue may be cells derived from the human heart, stomach, large intestine, small intestine, lung, liver, kidney, or uterus. In addition, cells derived from human tissue may be human-derived immortalized cell lines.

[0014] Stem cells may be mesenchymal stem cells, hematopoietic stem cells, neural stem cells, embryonic stem cells, or induced pluripotent stem cells. Specifically, the stem cells may be mesenchymal stem cells.

[0015] As used herein, the term “mesenchymal stem cells (MSCs)” refers to stem cells that differentiate from the mesoderm formed by the division of a fertilized egg and are present in the stroma of cartilage, bone tissue, adipose tissue, bone marrow, etc., and may include mesenchymal stem cells of mammals, including humans. Mesenchymal stem cells maintain stem cell properties and self-regeneration, and have the ability to differentiate into various cells, including chondrocytes, osteoblasts, muscle cells, and adipocytes. They can be extracted from umbilical cord, bone marrow, adipose tissue, umbilical cord blood, synovial membrane, bone tissue (cancellous bone), muscle, infrapatellar fat pad, peripheral blood, liver, teeth, hair follicles, etc. Mesenchymal stem cells are known to have immunomodulatory capabilities that suppress the activity and proliferation of T cells and B cells, inhibit the activity of natural killer cells, and regulate the function of dendritic cells and macrophages, and are therefore cells that can be transplanted allogeneically and xenotransplanted.

[0016] Mesenchymal stem cells may originate from, but are not limited to, the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, tooth, hair follicle cells, or placenta, or may be differentiated from induced pluripotent stem cells.

[0017] As used herein, the term “derived cells,” such as “derived mesenchymal stem cells,” refers to cells substantially isolated from the tissue from which they originate, or cells differentiated into induced stem cells.

[0018] As used herein, the term “isolated extracellular vesicles” refers to extracellular vesicles substantially isolated from the cells from which the extracellular vesicles originate, such as mesenchymal stem cells. Preferably, the extracellular vesicles can be isolated from the culture medium of the mesenchymal stem cells. The culture medium used to culture the cells may be any conventional medium known in the art to be suitable for cell culture. The medium may contain serum or may be a serum-free medium. Preferably, the medium may be a serum-free medium.

[0019] As used herein, the term “extracellular vesicle (EV)” refers to nano-sized particles (approximately 50 nm to approximately 1,000 nm) naturally secreted in living cells and enclosed in a lipid bilayer. Extracellular vesicles are known to be composed of biologically active substances such as DNA, RNA, and proteins, and to play a role in intercellular communication. In the present invention, extracellular vesicles may include vesicles having a composition similar to that of extracellular vesicles (exosomes, microvesicles, polyvesicles, and extracellular vesicle-like vesicles).

[0020] In the present invention, the extracellular vesicle may contain one overexpressed microRNA selected from the group consisting of hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-let-7f-5p, and combinations thereof.

[0021] As used herein, the term "microRNA (miRNA)" refers to small RNA molecules that regulate gene expression in living organisms. MicroRNAs are small RNA molecules containing 20 to 25 nucleotides that can play a crucial role in regulating gene expression by inhibiting target mRNA translation through complementary base pairing with the target mRNA 3'UTR (untranslated region). MicroRNAs play important roles in cellular functions, including proliferation, differentiation, and apoptosis. MicroRNAs are evolutionarily conserved regulatory molecules present in all animals. Some microRNAs are known to regulate gene expression through epigenetic regulatory mechanisms (such as histone modification and DNA methylation) associated with their promoter regions.

[0022] In one embodiment, the extracellular vesicle may contain overexpressed hsa-miR-148a-3p. In one embodiment, the extracellular vesicle may contain overexpressed hsa-miR-100-5p. In one embodiment, the extracellular vesicle may contain overexpressed hsa-miR-143-3p. In one embodiment, the extracellular vesicle may contain overexpressed hsa-let-7a-5p. In one embodiment, the extracellular vesicle may contain overexpressed hsa-let-7f-5p.

[0023] In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p and hsa-miR-100-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p and hsa-miR-143-3p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p and hsa-let-7a-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-100-5p and hsa-miR-143-3p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-100-5p and hsa-let-7a-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-100-5p and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-143-3p and hsa-let-7a-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-143-3p and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-let-7a-5p and hsa-let-7f-5p.

[0024] In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-100-5p, and hsa-miR-143-3p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-100-5p, and hsa-let-7a-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-100-5p, and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-143-3p, and hsa-let-7a-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-143-3p, and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-let-7a-5p, and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-100-5p, hsa-miR-143-3p, and hsa-let-7a-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-100-5p, hsa-miR-143-3p, and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-100-5p, hsa-let-7a-5p, and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-143-3p, hsa-let-7a-5p, and hsa-let-7f-5p.

[0025] In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, and hsa-let-7a-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-143-3p, hsa-let-7a-5p, and hsa-let-7f-5p. In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-100-5p, hsa-miR-143-3p, hsa-let-7a-5p, and hsa-let-7f-5p.

[0026] In one embodiment, the extracellular vesicles may contain overexpressed hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, hsa-let-7a-5p, and hsa-let-7f-5p.

[0027] In this case, the extracellular vesicles may contain hsa-miR-148a-3p at a concentration of approximately 1% to 20% of the total microRNA content of the extracellular vesicles. Specifically, the extracellular vesicles may contain approximately 1% to 20%, approximately 5% to 18%, or approximately 10% to 16%. Preferably, the extracellular vesicles may contain approximately 15.1% hsa-miR-148a-3p.

[0028] Extracellular vesicles may contain hsa-miR-100-5p at a concentration of approximately 1% to 20% of their total microRNA content. Specifically, extracellular vesicles may contain approximately 1% to 20%, 5% to 17%, or 10% to 14% of hsa-miR-100-5p. Preferably, extracellular vesicles may contain approximately 11% hsa-miR-100-5p.

[0029] Extracellular vesicles may contain hsa-miR-143-3p at a concentration of approximately 0.1% to 10% of their total microRNA content. Specifically, extracellular vesicles may contain approximately 0.1% to 10%, 0.5% to 8%, or 1% to 6% of hsa-miR-143-3p. Preferably, extracellular vesicles may contain approximately 4.9% hsa-miR-143-3p.

[0030] Extracellular vesicles may contain hsa-let-7a-5p at a concentration of approximately 0.1% to 10% of the total microRNA content of the extracellular vesicles. Specifically, extracellular vesicles may contain approximately 0.1% to 10%, approximately 0.5% to 8%, or approximately 1% to 6%. Preferably, extracellular vesicles may contain approximately 4.6% hsa-let-7a-5p.

[0031] Extracellular vesicles may contain hsa-let-7f-5p at a concentration of approximately 0.1% to 10% of the total microRNA content of the extracellular vesicles. Specifically, extracellular vesicles may contain approximately 0.1% to 10%, approximately 0.5% to 8%, or approximately 1% to 6%. Preferably, extracellular vesicles may contain approximately 3.8% hsa-let-7f-5p.

[0032] In this case, microRNA may possess fibrosis-inhibiting activity.

[0033] In one embodiment of the present invention, when lung epithelial cells or lung fibroblasts overexpressing microRNA were treated with TGF-β1, it was confirmed that the expression of genes and proteins related to epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transition (FMT) was reduced (Figures 15a, 15b, and 16-18).

[0034] In addition, the extracellular vesicles include hsa-miR-151a-3p, hsa-let-7b-5p, hsa-miR-21-5p, hsa-miR-10a-5p, hsa-miR-92a-3p, hsa-miR-486-5p, hsa-miR-99b-5p, hsa-let-7i-5p, hsa-miR-320a, hsa-miR-409-3p, hsa-miR-10b-5p, and hsa-miR-1 27-3p, hsa-miR-26a-5p, hsa-miR-221-3p, hsa-let-7e-5p, hsa-miR-493-5p, hsa-miR-25-3p, hsa-miR-574 -3p, hsa-miR-423-5p, hsa-miR-122-5p, hsa-miR-382-5p, hsa-miR-155-5p, hsa-miR-451a, hsa-miR-30a-3p , hsa-miR-28-3p, hsa-let-7g-5p, hsa-miR-379-5p, hsa-miR-23a-3p, hsa-miR-485-5p, hsa-miR-30a-5p, h sa-miR-30d-5p, hsa-miR-423-3p, hsa-miR-99a-5p, hsa-let-7c-5p, hsa-miR-92b-3p, hsa-miR-323a-3p, hs It may further contain any one expressed microRNA selected from the group consisting of a-miR-574-5p, hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-140-3p, hsa-miR-370-3p, hsa-miR-196a-5p, hsa-miR-654-5p, hsa-miR-184, hsa-miR-181a-2-3p, and combinations thereof.

[0035] The particle size of extracellular vesicles can range from approximately 10 nm to approximately 1,000 nm. Specifically, the particle size of extracellular vesicles can range from approximately 10 nm to approximately 1,000 nm, approximately 20 nm to approximately 900 nm, approximately 30 nm to approximately 800 nm, approximately 40 nm to approximately 700 nm, approximately 50 nm to approximately 600 nm, or approximately 60 nm to approximately 500 nm. Pharmaceutical composition In another embodiment of the present invention, a pharmaceutical composition for preventing or treating fibrosis is provided, comprising an extracellular vesicle as an active ingredient. The extracellular vesicle is the same as that described above.

[0036] As used herein, the term “fibrosis” refers to the formation of excessive fibrous connective tissue in an organ or tissue. This excessive fibrous connective tissue is distinguished from the normal fibrous tissue that constitutes the organ or tissue. Fibrosis can be understood as a fatal disease characterized by the excessive accumulation of extracellular matrix, such as fibronectin and collagen, by fibroblasts, leading to a permanent loss of human tissue function due to hardening of organ tissue.

[0037] Fibrosis can occur in one or more organs selected from the group consisting of, for example, the kidneys, liver, lungs, skin, heart, pancreas, urinary system, reproductive system, sweat glands, nerves, brain, bone marrow, muscles, and joints.

[0038] In the present invention, fibrosis may be hepatic fibrosis, pulmonary fibrosis, cutaneous fibrosis, articular fibrosis, neurofibrosis, pancreatic fibrosis, renal fibrosis, muscular fibrosis, or peritoneal fibrosis. Specifically, fibrosis may be pulmonary fibrosis, but is not limited thereto.

[0039] As used herein, the term “pulmonary fibrosis” refers to the development of scar tissue resulting from the formation or development (fibrosis) of excessive fibrous connective tissue in the lungs. Specifically, pulmonary fibrosis is a chronic disease that causes swelling and scarring of the alveoli and interstitial tissue of the lungs. This scar tissue replaces healthy tissue and causes inflammation, and chronic inflammation can be identified as a precursor to fibrosis. This damage to lung tissue can harden the lungs and make it difficult for the subject to breathe on their own.

[0040] Specifically, pulmonary fibrosis can include, but is not limited to, one or more conditions selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced lung injury, nonspecific interstitial pneumonia, acute interstitial pneumonia, idiopathic organizing pneumonia, interstitial lung disease with respiratory bronchiolitis, desquamative interstitial pneumonia, lymphocytic interstitial pneumonia, interstitial pulmonary fibrosis and diffuse pulmonary fibrosis, pulmonary edema, cystic fibrosis, and pulmonary fibrosis caused by metabolic diseases.

[0041] In the present invention, the extracellular vesicles, which are the active ingredient of the pharmaceutical composition, may be included in any amount (effective amount) depending on the purpose, formulation, and mixing purpose, as long as they can exhibit fibrosis inhibitory activity. A typical effective amount of extracellular vesicles is determined in the range of 0.001 wt% to 20.0 wt% based on the total weight of the composition. Here, "effective amount" refers to the amount of the active ingredient that can induce a fibrosis inhibitory effect. Such an effective amount can be experimentally determined within the range of the usual ability of those skilled in the art.

[0042] As used herein, the term “treatment” may be used to mean both therapeutic and preventive treatments. In this context, prevention may be used to mean alleviating or reducing a pathological condition or disease in a subject. In one embodiment, the term “treatment” includes all applications or any form of a medicament for treating a disease in a mammal, including humans. In addition, the terms include inhibiting or slowing a disease or its progression; restoring or repairing damaged or missing functions, thereby partially or completely alleviating a disease; stimulating inefficient processes; or alleviating a serious disease.

[0043] As used herein, the term “effectiveness” may be determined by one or more parameters, such as survival or disease-free survival over a period of time, such as one year, five years, or ten years. In addition, parameters may include suppression of the size of at least one tumor in the subject.

[0044] Pharmacokinetic parameters such as bioavailability, as well as underlying parameters such as clearance rates, can also affect efficacy. Therefore, "improved efficacy" (e.g., improved efficacy) may be due to improvements in pharmacokinetic parameters and efficacy, and can be measured by comparing clearance rates and tumor growth in test animals or human subjects, or by comparing parameters such as survival time, recurrence rate, or disease-free survival time.

[0045] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, transdermally, nasally, or intratracheally), preferably parenterally, depending on the method of administration. The dosage may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration of administration, but can be appropriately selected by those skilled in the art.

[0046] The pharmaceutical compositions according to the present invention are administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk applicable to the medical treatment. The level of the effective dose may be determined according to factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, drugs used concurrently, and other factors well known in the medical field. The compositions according to the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or concurrently with conventional therapeutic agents, and can be administered in single doses or multiple doses. By considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this amount can be easily determined by those skilled in the art.

[0047] Specifically, the preferred dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, sex, and age, the severity of the patient's condition, and the route of administration. Typically, the dosage may range from about 0.001 mg to about 150 mg per kg of body weight, preferably from about 0.01 mg to about 100 mg per kg of body weight, and may be administered daily, every other day, or in 1 to 3 divided doses per day. However, the above dosage may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., and therefore should not be construed as limiting the scope of the present invention in any way.

[0048] The target population to which the pharmaceutical composition may be applied (formulated) may be mammals, preferably humans. In addition to the active ingredient, the pharmaceutical composition of the present invention may further contain any compound or natural extract that has already been proven safe and is known to have fibrosis inhibitory activity in order to increase and / or enhance antifibrotic activity.

[0049] The pharmaceutical composition may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any carrier, as long as it is a non-toxic substance suitable for delivery to the patient. Distilled water, alcohol, fats, waxes, and inert solids may be included as carriers. In addition, a pharmaceutically acceptable adjuvant (buffer, dispersant) may be included in the pharmaceutical composition.

[0050] Specifically, the pharmaceutical composition can be prepared by conventional methods known in the art as a parenteral formulation depending on the route of administration, comprising an active ingredient in addition to a pharmaceutically acceptable carrier. Here, "pharmaceutically acceptable" means that it does not inhibit the activity of the active ingredient and does not have toxicity exceeding that which the target of application (prescription) can tolerate.

[0051] When a pharmaceutical composition is prepared as a parenteral formulation, it can be formulated in the form of an injectable, transdermal preparation, nasal inhalant, or suppository, in combination with a suitable carrier, using methods known in the art. When a pharmaceutical composition is formulated as an injectable, suitable carriers may include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, PBS (phosphate-buffered saline) containing triethanolamine, or an isotonic solution such as sterile water for injection or 5% dextrose. Methods for formulating pharmaceutical compositions are well known in the art, and specifically, refer to Remington's Pharmaceutical Sciences (19th edition, 1995), which is incorporated herein by reference.

[0052] In another aspect of the present invention, the use of an extracellular vesicle or a pharmaceutical composition containing an extracellular vesicle as an active ingredient for the prevention or treatment of fibrosis is provided.

[0053] In another embodiment of the present invention, a method for preventing or treating fibrosis is provided, comprising the step of administering to a subject extracellular vesicles or a pharmaceutical composition containing extracellular vesicles as an active ingredient, wherein the extracellular vesicles, pharmaceutical composition, fibrosis, prevention, treatment, and administration are the same as those described above. The subject may be a mammal, preferably a human. In addition, the subject may be a patient suffering from the disease or a subject at high risk of suffering from the disease.

[0054] In addition, the route of administration, dosage, and frequency of administration may vary depending on the patient's condition and the presence or absence of side effects, and it may be administered to the target by various means and in various amounts. The optimal method of administration, dosage, and frequency of administration can be selected within a reasonable range by those skilled in the art.

[0055] Specifically, the preferred dose of extracellular vesicles or pharmaceutical composition may vary depending on the patient's condition, weight, sex, and age, the severity of the patient's condition, and the route of administration. Typically, the dose may range from about 0.001 mg to about 150 mg per kg of body weight, preferably from about 0.01 mg to about 100 mg per kg of body weight, and may be administered daily, every other day, or in 1 to 3 divided doses per day. However, the above dose may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., and therefore should not be construed as limiting the scope of the present invention in any way. [Examples]

[0056] The present invention will be described in more detail below by the following examples. However, these examples are intended to illustrate the present invention only, and the scope of the invention is not limited to these examples.

[0057] Example 1. Isolation of extracellular vesicles from mesenchymal stem cells Extracellular vesicles were isolated from umbilical cord-derived mesenchymal stem cells using the process shown in Figure 2.

[0058] Specifically, human umbilical cord-derived mesenchymal stem cells were cultured in CSBM-A06 (Sigma Aldrich) culture medium containing 10% fetal bovine serum (Gibco, 16000-044), 1% penicillin / streptomycin (Gibco, 15140-122), and 1% glutamax (Gibco, 236320). After the culture was complete, the mesenchymal stem cells were washed with Dulbecco's phosphate-buffered saline (DPBS, Gibco, 14040-177) and replaced with a medium containing 1% glutamax. The cells were then cultured for 48 hours, and the cell culture medium (culture supernatant) was collected. The reason for using serum-free medium was to prevent mixing of extracellular vesicles derived from fetal bovine serum (FBS) with extracellular vesicles secreted by stem cells, given that fetal bovine serum (FBS) contains a significant amount of extracellular vesicles derived from FBS.

[0059] The collected cell culture medium was centrifuged at 300 × g for 10 minutes to remove all remaining cells. The resulting supernatant was centrifuged again at 2,000 × g for 20 minutes to remove cell fragments and filtered through a 0.2 μm filter. The filtrate was then centrifuged in an ultracentrifuge at 100,000 × g at 4°C for 70 minutes. After ultracentrifugation, the supernatant was removed, and the remaining extracellular vesicle pellet in the lower layer was suspended in DPBS and centrifuged again at 100,000 × g at 4°C for 70 minutes. The supernatant was removed, and the remaining extracellular vesicle pellet in the lower layer was diluted in physiological saline (DAI HAN PHARM CO., LTD.) and used in the following experiments.

[0060] Example 2. Comparison of the physical and biochemical properties of extracellular vesicles derived from mesenchymal stem cells. The physical and biochemical properties of extracellular vesicles (hMSC-EVs) derived from human mesenchymal stem cells were confirmed using the same method as in Example 1 above, employing a nanoparticle tracking analyzer, cryogenic transmission electron microscope, Western blotting, and flow cytometry.

[0061] First, the size and number of extracellular vesicles were measured using a nanoparticle tracking analyzer. As shown in Figure 3, it was confirmed that the extracellular vesicles had an average size of 200 nm or less.

[0062] In addition, as shown in Figure 4, when the cell membrane was observed using a cryogenic transmission electron microscope, a biphospholipid membrane structure was observed in extracellular vesicles.

[0063] The expression of extracellular vesicle marker proteins was confirmed using Western blotting. The antibodies used were anti-CD9 antibody (Santa Cruz, sc-13118), anti-CD63 antibody (Santa Cruz, sc-5275), anti-CD81 antibody (Santa Cruz, sc-7637), anti-GRP94 antibody (Santa Cruz, sc-32249), anti-β-actin antibody (Santa Cruz, sc-47778), or anti-cytochrome C antibody (Santa Cruz, sc-13156).

[0064] As a result, as shown in Figure 5, the expression of CD9, CD63, and CD81, which are marker proteins for extracellular vesicles (exosomes), was confirmed in the extracellular vesicle sample. On the other hand, GRP94, cytochrome C, and β-actin (for purity confirmation), which are proteins known not to be expressed in extracellular vesicles, were expressed only in the cell sample.

[0065] In addition, the phenotype of extracellular vesicles was reconfirmed by flow cytometry. The antibodies used were anti-CD9-PE antibody (BD, 555372), anti-CD63-PE antibody (BD, 556020), anti-CD81-APC antibody (BD, 551112), anti-GM130-PE antibody (Santa Cruz, sc55590), and anti-calnexin-FITC antibody (Santa Cruz, sc-23954). PE isotype IgG1 (BD Biosciences, 555749), APC isotype IgG1 (BD Biosciences, 555751), or FITC isotype IgG1 (BD Biosciences, 555748) were used as control antibodies, respectively.

[0066] As a result, as shown in Figure 6, GM130 and calnexin, proteins used to confirm purity, were expressed at less than 10%, while the extracellular vesicle marker proteins CD9, CD63, and CD81 were confirmed to be expressed at more than 90% in the extracellular vesicle sample.

[0067] Example 3. Isolation of extracellular vesicles from mesenchymal stem cells In order to evaluate the unique factors of the extracellular vesicles (hMSCs) derived from human mesenchymal stem cells isolated in Example 1 above, small RNA sequencing was performed to analyze the types and expression levels of microRNAs.

[0068] Specifically, RNA QC testing was performed on extracellular vesicles using the Bioanalyzer 2100 system. Total RNA was extracted according to the manufacturer's protocol, and cDNA libraries were constructed from samples that passed the QC testing and analyzed using next-generation sequencing (NGS) technology. The results were then used to perform a comparative analysis of the overall microRNA profile.

[0069] As a result, as shown in Figures 7a and 7b, it was confirmed that the extracellular vesicles contained 50 types of microRNAs. In particular, it was confirmed that large amounts of miR-148a-3p, let-7a-5p, let-7b-5p, miR-100-5p, miR-143-3p, and let-7f-5p were present among the microRNAs.

[0070] Example 4. In vitro evaluation of the therapeutic efficacy of extracellular vesicles derived from human mesenchymal stem cells for pulmonary fibrosis. Example 4.1. Analysis of gene expression of fibrosis-related factors To confirm the inhibitory effect of extracellular vesicles obtained according to the method described in Example 1 above on pulmonary fibrosis, an in vitro model similar to the pathogenesis of EMT and FMT was constructed using lung epithelial cells, and the therapeutic efficacy against pulmonary fibrosis was evaluated.

[0071] Specifically, human lung epithelial cells (A549 cells) or human lung fibroblasts (NHLF cells) are placed in 2 × 10⁶ well plates. 5 The cells were seeded at a cell / well concentration and cultured for 24 hours under conditions of 5% CO2 and 37°C. Then, the cell culture medium was removed, and the cells were washed twice with PBS. TGF-β1 (transforming growth factor β1) (10 ng / mL) and hMSC-EV (1 × 10⁻¹⁶) prepared according to the method in Example 1 were added. 8 particles / mL, 1×10 9The cells were treated with (particles / mL). After a further 48 hours of incubation, the cell culture medium was removed and washed with PBS. RNA was extracted from the cells using the Easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology) according to the manufacturer's protocol, and the RNA was quantified using Nanodrop. cDNA synthesis was performed in a nucleic acid amplifier using the above RNA (1 μg) as a template and Maxime RT premix (oligo dT primer) according to the manufacturer's protocol. The nucleic acid amplifier was operated at 45°C for 60 minutes and at 95°C for 5 minutes. After the reaction was complete, the obtained cDNA was diluted to 1 / 5 in DEPC-treated water (80 μL) and used as the sample cDNA. The gene expression levels of each sample were analyzed by q-PCR using the respective primers for TGF-β1, fibronectin, α-SMA (smooth muscle actin), CTGF (connective tissue growth factor), COL3A1 (collagen type III alpha 1 chain), E-cadherin, and GAPDH. The primer sequences used are shown in Table 1. [Table 1]

[0072] As a result, as shown in Figure 8, it was confirmed that the gene expression of fibrosis-related factors was significantly reduced in the TGF-β1+hMSC-EV treatment group compared to the TGF-β1-only treatment group.

[0073] Example 4.2. Analysis of wound closure effect To confirm the epithelial-mesenchymal transition (EMT) inhibitory effect of extracellular vesicles obtained according to the method described in Example 1 above, wound closure experiments were performed.

[0074] Specifically, A549 cells were placed in 6 x 10⁶ well plates. 5Seeded at the cell / well concentration and then cultured for 24 hours under the conditions of 5% CO2 and 37 °C. After 24 hours, the monolayer of cells was scratched using a 200 μL pipette tip to form a scratch. The cell culture medium was removed, and then the cells were washed twice with PBS and treated with TGF-β1 (10 ng / mL) and hMSC-EV (1×10 8 particles / mL, 1×10 9 particles / mL) prepared according to the method described in Example 1, followed by further incubation for 48 hours. The cells were observed under a microscope and imaged during the incubation period (0, 24, and 48 hours).

[0075] As a result, as shown in Figure 9, it was confirmed that wound closure was delayed in a concentration- and treatment time-dependent manner in the TGF-β1 + hMSC-EV treatment group compared to the TGF-β1-only treatment group.

[0076] Example 4.3. Analysis of cell invasion inhibitory effect The inhibitory effect of extracellular vesicles obtained according to the method described in Example 1 above on cell invasion (migration and invasion) was confirmed.

[0077] Specifically, a 0.8 μm pore size Transwell (Corning) was used to confirm invasion and migration. The cell culture medium used in the invasion assay (migration assay) was prepared by treating serum-free DMEM / high glucose medium with TGF-β1 (10 ng / mL) and hMSC-EV (1×10 8 particles / mL, 1×10 9 particles / mL) according to the method described in Example 1. A549 cells were seeded at 5×10 4Cells were suspended at a concentration of 300 μL and added to the upper chamber of the Transwell. 700 μL of DMEM / high-glucose medium supplemented with 10% FBS was added to the lower chamber, and then the two chambers were combined. The cells were cultured for 24 hours under conditions of 5% CO2 and 37°C, fixed with 70% ethanol, and then stained with crystal violet solution. The membrane of the upper chamber was then wiped with a cotton swab and observed under a microscope to count the number of cells within the membrane.

[0078] The upper chamber of the Transwell was coated with 100 μL (20 μg / well) of Matrigel, and then a migration assay (invasion assay) was performed using the same method as the invasion assay.

[0079] As a result, as shown in Figure 10, it was confirmed that cell migration and invasion were reduced in the TGF-β+hMSC-EV treated group compared to the TGF-β-only treated group.

[0080] Example 4.4. Analysis of the cell invasion inhibitory effect To confirm the mechanism of action of extracellular vesicles obtained according to the method in Example 1 in inhibiting epithelial-mesenchymal transition (EMT), the expression of fibrosis-related proteins was confirmed using Western blotting.

[0081] Specifically, A549 cells were placed in 2 × 10⁶ well plates. 5 Cells were seeded at a concentration of cells / well and then cultured for 24 hours under conditions of 5% CO2 and 37°C. The culture medium was removed and the cells were washed twice with PBS. The cells were then treated with TGF-β1 (10 ng / mL) and hMSC-EV (1 × 10⁻¹⁶) prepared according to the method described in Example 1. 8 particles / mL, 1×10 9The cells were treated with (particles / mL). After a further 48 hours of incubation, the culture medium was removed and washed with PBS. The cells were scraped using a scraper and collected in a 15 mL tube. After centrifugation, the supernatant was removed, and the cell pellet was resuspended with RIPA solution containing a protease inhibitor. The suspension was vortexed on ice for 30 minutes (vortexing every 10 minutes), followed by centrifugation (13,000 rpm, 30 minutes, 4°C). The supernatant was collected, and the protein was quantified using the Bradford assay (BioRad).

[0082] The protein (30 μg) obtained through the above process was subjected to electrophoresis on a 10% SDS-PAGE gel and transferred to a PVDF (polyvinylidene fluoride) membrane. The membrane was then blocked at room temperature for 1 hour using blocking buffer (TBST buffer containing 5% skim milk powder), and subsequently reacted with primary antibody overnight at 4°C. The primary antibodies used were anti-TGFβ1 antibody (Abcam, ab92486), anti-fibronectin antibody (Abcam, ab2413), anti-COL1A1 antibody (Abcam, ab34710), anti-E-cadherin antibody (Cell Signaling, 3195S), anti-p-Smad2 antibody (Cell Signaling, 3108T), anti-Smad2 antibody (Cell Signaling, 5339T), or anti-β-actin antibody (Santa Cruz, sc-47778).

[0083] After the primary antibody reaction, washing was performed three times using TBST (Tris-buffered saline and Tween 20) solution, followed by reaction with the secondary antibody at room temperature for 1 hour. The secondary antibodies used were either anti-mouse antibody (Cell Signaling, 7076s) or anti-rabbit antibody (Cell Signaling, 7074s). Subsequently, protein expression was confirmed by chemiluminescence imaging using ECL solution.

[0084] In this study, β-actin was used as an indicator to confirm the protein content of each sample to which equal amounts were added. TGF-β1 was used as an EMT inducer, and TGF-β1, fibronectin, and COL1A1 were used as fibrosis markers. In addition, E-cadherin was used as an epithelial cell marker, and p-Smad2 and Smad2 were used as downstream signaling proteins involved in TGF-β-induced EMT.

[0085] As a result, as shown in Figure 11, it was confirmed that treatment with TGF-β1 increased the expression of mesenchymal cell markers fibronectin, COL1A1, p-Smad2, and TGF-β1. On the other hand, it was confirmed that treatment with extracellular vesicles decreased the expression of mesenchymal cell markers increased by TGF-β1 and increased the expression of the epithelial cell marker E-cadherin.

[0086] Example 4.5. Analysis of Cell Immunofluorescence Staining To confirm the mechanism of action of the extracellular vesicles obtained according to the method in Example 1 above in inhibiting epithelial-mesenchymal transition (EMT), fibronectin expression was confirmed using cellular immunofluorescence staining.

[0087] Specifically, A549 cells were placed in 2 × 10⁶ well plates. 5 Cells were seeded at a concentration of cells / well and then cultured for 24 hours under conditions of 5% CO2 and 37°C. The culture medium was removed and the cells were washed twice with PBS. The cells were then treated with TGF-β1 (10 ng / mL) and hMSC-EV (1 × 10⁻¹⁶) according to the method described in Example 1. 8 particles / mL, 1×10 9The cells were treated with (particles / mL). After a further 48 hours of incubation, the culture medium was removed and washed with PBS. The cells were fixed with 4% paraformaldehyde for 15 minutes, and then washed three times with PBS. Blocking solution was added, and blocking was performed at 60 rpm for 30 minutes. The cells were then washed three times with PBS. The reaction with a primary antibody diluted in a ratio of 1:1000 was carried out overnight at 4°C. The primary antibody used was an anti-fibronectin antibody (Abcam, ab2413).

[0088] After the primary antibody reaction, the cells were washed three times with PBS and then reacted with a secondary antibody diluted in a 1:1000 ratio at room temperature for 1 hour in the dark. The secondary antibody used was an anti-rabbit antibody (Invitrogen, A11008). Subsequently, the cells were washed three times with PBS, and nuclear staining was performed by adding DAPI fluorescent agent (ThermoScientific, 62248) and reacting at room temperature for 5 minutes in the dark. After that, the cells were washed three times with PBS and observed under a fluorescence microscope.

[0089] As a result, as shown in Figure 12, it was confirmed that fibronectin expression was reduced in the TGF-β1+hMSC-EV treated group compared to the TGF-β1 treated group.

[0090] Example 5. In vitro evaluation of the therapeutic efficacy of increased microRNA expression for pulmonary fibrosis. Example 5.1. Production of lung epithelial cells and lung fibroblasts with increased microRNA expression. To confirm the therapeutic effect of increased microRNA expression in lung epithelial cells or lung fibroblasts on pulmonary fibrosis, microRNAs (miR-148a-3p (SEQ ID NO: 35), miR-100-5p (SEQ ID NO: 38), miR-143-3p (SEQ ID NO: 39), let-7a-5p (SEQ ID NO: 36), let-7b-5p (SEQ ID NO: 37), or let-7f-5p (SEQ ID NO: 40)) were first transduced into A549 cells (Figure 13) and lung fibroblasts (NHLF) (Figure 14). Subsequently, gene expression analysis was performed to confirm whether or not the transduced microRNAs were overexpressed.

[0091] Specifically, human lung epithelial cell line A549 cells, or normal human lung fibroblasts (NHLF cells), are placed in a 6-well plate in a 1 × 10⁶ layer. 5 Cells were seeded at a concentration of cells / well and then cultured for 24 hours under conditions of 5% CO2 and 37°C. Afterward, the culture medium was removed from each well, and the cells were washed twice with PBS. Next, lentiviruses (VectorBuilder) containing each microRNA to be overexpressed were diluted in serum-free medium and applied to each well at a MOI of 10. After 6 hours, the culture medium was removed from each well, and the cells were washed twice with PBS. The cells were then cultured in serum-supplemented medium at 37°C and 5% CO2 for 48 hours.

[0092] Subsequently, the cell culture medium was removed from each well, the cells were washed twice with PBS, and then RNA was extracted. RNA was extracted using the miRNeasy Tissue / Cell Modification Microkit (QIAGEN) according to the manufacturer's protocol, and the extracted RNA was quantified using Nanodrop. cDNA synthesis was performed using 200 ng of RNA in a nucleic acid amplification device with the miRCURY LNA RT Kit (QIAGEN) according to the manufacturer's protocol (reaction at 40°C for 60 minutes, then at 95°C for 5 minutes). The synthesized cDNA, as described above, was diluted to a ratio of 1 / 60 and used as template cDNA. Gene expression was analyzed using a real-time nucleic acid amplification device with the miRCURY LNA SYBR Green PCR kit (QIAGEN) along with miR-148a-3p, let-7a-5p, let-7b-5p, miR-100-5p, miR-143-3p, or let-7f-5p, and U6 primer (QIAGEN).

[0093] As a result, as shown in Figures 13 and 14, it was confirmed that the expression of each transfected microRNA increased in A549 cells and NHLF cells that overexpressed each microRNA.

[0094] Example 5.2. In vitro confirmation of changes in fibrosis-related gene expression due to increased microRNA expression. A549 cells or NHLF cells overexpressing each microRNA were treated with TGF-β1 (10 ng / mL) in the same manner as in Example 5.1 above. After 24 hours, gene expression of TGF-β1, fibronectin, α-SMA, vimentin, CTGF (connective tissue growth factor), E-cadherin, or U6 was confirmed by q-PCR using the same method as in Example 4.1 above. The primer sequences used are shown in Table 1 above.

[0095] As a result, in the case of A549 cells, as shown in Figures 15a and 15b, the expression of TGF-β1, vimentin, and CTGF, which were increased by TGF-β1 treatment, decreased in miR-148a-3p overexpressing cells compared to the scrambled treatment group, while E-cadherin expression increased. α-SMA expression decreased in miR-143-3p overexpressing cells, TGF-β1 expression decreased in let-7a-5p overexpressing cells, and TGF-β1 and vimentin expression decreased in let-7b-5p overexpressing cells.

[0096] In NHLF cells, as shown in Figure 16, the expression of TGF-β1, vimentin, fibronectin, and COL3A1, which was increased by TGF-β1 treatment, was decreased in miR-148a-3p overexpressing cells compared to the scrambled treatment group, and the expression of TGF-β1, α-SMA, vimentin, and COL3A1 was decreased in miR-100-5p overexpressing cells. In addition, the expression of TGF-β1, α-SMA, vimentin, CTGF, fibronectin, and COL3A1 was decreased in miR-143-3p overexpressing cells, and the expression of vimentin and COL3A1 was decreased in let-7b-5p overexpressing cells. The expression of fibronectin and COL3A1 was decreased in let-7f-5p overexpressing cells.

[0097] Example 5.3. In vitro confirmation of changes in fibrosis-related protein expression due to increased microRNA expression. A549 cells or NHLF cells overexpressing each microRNA were treated with TGF-β1 (10 ng / mL) in the same manner as in Example 5.1 above. After 24 hours, the medium in each well was removed, washed with PBS, and each well was treated again with PBS (1 mL). The cells were collected using a scraper. Western blotting was performed on the cells obtained as described above, using the same method as in Example 4.4 above.

[0098] The primary antibodies used were anti-TGFβ1 antibody (Abcam, ab92486), anti-vimentin antibody (Cell Signaling, 5741S), anti-CTGF antibody (Abcam, ab6992), anti-E-cadherin antibody (Cell Signaling, 3195S), anti-p-Smad2 antibody (Cell Signaling, 3108T), anti-Smad2 antibody (Cell Signaling, 5339T), or anti-β-actin antibody (Santa Cruz, sc-47778). TGF-β1 was used as an EMT inducer, and TGF-β1, α-SMA, CTGF, E-cadherin, and vimentin were used as evaluation markers. In addition, p-Smad2 was used to confirm whether TGF-β inhibited the downstream EMT mechanism.

[0099] As a result, in the case of A549 cells, as shown in Figure 17 (A549), it was confirmed that the protein expression of TGF-β1 and vimentin was decreased in cells overexpressing miR-148a-3p, let-7a-5p, let-7b-5p, miR-100-5p, or miR-143-3p compared to the scrambled treatment group. In addition, in the case of NHLF cells, as shown in Figure 18, it was confirmed that the protein expression of TGF-β1 and α-SMA was decreased in cells overexpressing miR-148a-3p, miR-100-5p, or miR-143-3p compared to the scrambled treatment group. On the other hand, it was confirmed that the expression of SMAD7, which can inhibit TGF-β expression, was increased.

[0100] Example 6. In vivo evaluation of the therapeutic efficacy of extracellular vesicles derived from human mesenchymal stem cells for pulmonary fibrosis. Example 6.1. Evaluation of therapeutic efficacy against pulmonary fibrosis in an animal model of pulmonary fibrosis induced by intraperitoneal administration of bleomycin. Example 6.1.1. Construction of an animal model of pulmonary fibrosis and administration of extracellular vesicles. To confirm the therapeutic effect of extracellular vesicles obtained according to the method described in Example 1 above on pulmonary fibrosis, an animal model of pulmonary fibrosis was created and the therapeutic effect of extracellular vesicles on pulmonary fibrosis was evaluated.

[0101] Specifically, as shown in the schematic diagram of the animal experiment in Figure 19, a pulmonary fibrosis model was created by intraperitoneally administering bleomycin (BLM), known to induce pulmonary fibrosis, at a concentration of 25 units / kg (100 μL) once daily for a total of 6 days to 8-week-old male C57BL / 6 mice (25 g). The extracellular vesicles used in the experiment were 2 × 10⁶. 10 To enable administration at a particle / mouse concentration, extracellular vesicles obtained according to the method described in Example 1 above were prepared by dispersing them in physiological saline solution, thereby preparing an injectable formulation.

[0102] Extracellular vesicles (2 × 10) prepared as described above 10 Particles (for mice) were administered intravenously to pulmonary fibrosis model mice as a single dose of 100 μL per mouse (16 days after the start of bleomycin administration). During the administration and observation period, systemic symptoms and body weight were observed and recorded once daily for 8 days for each animal. Eight days after administration of extracellular vesicles (24 days after the start of bleomycin administration), lung tissue was collected from the mice and subjected to soluble collagen assay and Masson's trichrome staining to confirm the therapeutic effect of extracellular vesicles on pulmonary fibrosis. Example 6.1.2. Confirmation of changes in gene expression of fibrosis-related factors in lung tissue. Lung tissue was obtained from the experimental mice described in Example 6.1.1 above, and the gene expression of fibrosis-related factors in the lung tissue was confirmed using q-PCR.

[0103] Specifically, the right middle lobe of lung tissue from the experimental mice described in Example 6.1.1 above was extracted and pulverized using a homogenizer. Trizol solution (1 mL) was added to the pulverized tissue sample and reacted overnight at -80°C. RNA was then extracted using an RNA extraction kit. After measuring the RNA concentration, cDNA was synthesized according to the manufacturer's protocol. Gene expression levels in each sample were confirmed by q-PCR using primers for COL1A1 (collagen type I alpha 1 chain), COL3A1, CTGF, elastin, and RPL13A (ribosomal protein L13a). The primer sequences used are shown in Table 1.

[0104] As a result, as shown in Figure 20, it was confirmed that the gene expression of COL1A1, COL3A1, CTGF, and elastin increased with bleomycin (BLM) administration, and that the increased gene expression of the above genes decreased with BLM + hMSC-EV administration.

[0105] Example 6.1.3. Confirmation of collagen deposition in lung tissue using a soluble collagen assay. Lung tissue was obtained from the experimental mice described in Example 6.1.1 above, and the concentration of collagen present in the lung tissue was measured using a soluble collagen assay.

[0106] Specifically, the right middle lobe of the lung obtained from the experimental mouse in Example 6.1.1 above was extracted, and the extracted tissue was pulverized using a homogenizer. The pulverized tissue was treated with pepsin-ascorbic acid solution and reacted overnight at 4°C. Then, centrifugation was performed at 3,000 × g for 10 minutes, and the supernatant was collected. The supernatant (100 μL) was mixed with Sircol dye reagent (1 μL) and reacted at room temperature for 30 minutes with shaking. After the reaction was complete, centrifugation was performed at 13,000 × g for 10 minutes, the supernatant was removed, and acid salt washing reagent (750 μL) was added and mixed by vortexing. The mixture was again centrifugated at 13,000 × g for 10 minutes, the supernatant was removed, and alkaline reagent (1 μL) was added and mixed by vortexing. The mixture (200 μL) was transferred to a 96-well plate, and the absorbance (556 nm) was measured using a microplate reader.

[0107] As a result, as shown in Figure 21, bleomycin (BLM) administration increased collagen concentration in lung tissue, while BLM + hMSC-EV administration significantly decreased collagen concentration.

[0108] Example 6.1.4. Confirmation of collagen deposition in lung tissue using Masson's trichrome staining. Collagen deposition in lung tissue obtained from the experimental mice described in Example 6.1.1 above was confirmed using Masson's trichrome staining.

[0109] Specifically, the left lobe of the lung tissue obtained from the experimental mouse in Example 6.1.1 above was extracted and fixed in a 4% paraformaldehyde solution. The fixed mouse lung tissue was then cut into 4 μm thick paraffin tissue sections.

[0110] The paraffin was removed from the tissue sections, they were hydrated, and then washed with water. The washed tissue sections were treated with Bouin's solution, fixed again at 56°C for 1 hour, and then washed under running water for 5-10 minutes. Next, the tissue sections were stained with Weigert's iron hematoxylin solution for 10 minutes, washed under running water for 10 minutes, and then washed again with distilled water. Next, the tissue sections were stained with Biebrich's scarlet-acid fuchsin solution for 10-15 minutes, and then washed again with distilled water. The sections were reacted in phosphomolybdenum-phosphotungstic acid solution for 10-15 minutes, and then reacted in aniline blue solution for a further 5-10 minutes. The stained tissue sections were washed with distilled water and then reacted in 1% acetic acid solution for 2-5 minutes. Next, the sections were dehydrated in 95% ethanol, treated with xylene solution, mounted using mounting solution, and observed under a microscope.

[0111] As a result, as shown in Figure 22, stained collagen regions were observed over a wide area in the bleomycin (BLM) administration group. On the other hand, in the BLM + hMSC-EV administration group, the degree of collagen deposition in lung tissue was reduced to a level equivalent to that of the normal group.

[0112] Example 6.2. Evaluation of therapeutic efficacy against pulmonary fibrosis in an animal model of pulmonary fibrosis induced by intratracheal administration of bleomycin. Example 6.2.1. Construction of an animal model of pulmonary fibrosis and administration of extracellular vesicles. To confirm the therapeutic effect of extracellular vesicles obtained according to the method described in Example 1 above on pulmonary fibrosis, an animal model of pulmonary fibrosis was created, and the therapeutic effect of extracellular vesicles on pulmonary fibrosis was evaluated in the created animal model of pulmonary fibrosis.

[0113] Specifically, a pulmonary fibrosis model was created by intratracheally administering bleomycin (BLM), known to induce pulmonary fibrosis, at a concentration of 1 mg / kg (100 μL) once daily to 8-week-old male C57BL / 6N mice (Figure 23). The number of extracellular vesicles used in the experiment was 1 × 10⁶ per mouse.3 , 1 x 10 5 , or 1 × 10 7 The extracellular vesicles obtained according to the method described in Example 1 above were prepared by dispersing them in a saline solution so that they could be administered in doses of individual particles, thereby preparing the injectable formulation.

[0114] Extracellular vesicles (1 × 10) prepared as described above 3 , 1 x 10 5 , and 1 × 10 7 Particles (for mice) were administered intravenously to pulmonary fibrosis model mice as a single dose of 100 μL per mouse (8 days after the start of bleomycin administration). During the administration and observation period, systemic symptoms and body weight were observed and recorded once daily for each animal. On day 13 after administration of extracellular vesicles (21 days after the start of bleomycin administration), lung tissue was collected from the mice for morphological examination (Figure 24). The therapeutic effect of extracellular vesicles on pulmonary fibrosis was confirmed using the expression of fibrosis marker genes and proteins, as well as soluble collagen assays and Masson's trichrome staining.

[0115] Example 6.2.2. Confirmation of changes in gene expression of fibrosis-related factors in lung tissue Lung tissue was obtained from the experimental mice described in Example 6.2.1 above, and the gene expression of fibrosis-related factors in the lung tissue was confirmed using q-PCR.

[0116] Specifically, the right middle lobe of lung tissue from the experimental mice described in Example 6.2.1 above was extracted and pulverized using a homogenizer. Trizole solution (1 mL) was added to the pulverized tissue sample and reacted overnight at -80°C. RNA was then extracted using an RNA extraction kit. After measuring the RNA concentration, cDNA was synthesized according to the manufacturer's protocol. Gene expression levels in each sample were confirmed by q-PCR using primers for COL1A1 (collagen type I alpha 1 chain), α-SMA, Timp, and fibronectin. The primer sequences used are shown in Table 1.

[0117] As a result, as shown in Figure 25, it was confirmed that the gene expression of COL1A1 (collagen type I alpha 1 chain), Timp, and fibronectin increased with bleomycin (BLM) administration, and that the increased gene expression of the above genes decreased with hMSC-EV administration.

[0118] Example 6.2.3. Confirmation of fibrosis-related protein expression in lung tissue The right middle lobe of the lung obtained from the experimental mice in Example 6.2.1 described above was extracted and pulverized using RIPA buffer and a homogenizer. Pulverization was carried out at 2-8°C for 20 minutes, and the supernatant protein was isolated and used by centrifugation at 13,000 rpm for 15 minutes.

[0119] The protein samples were subjected to Western blotting using the same method as in Example 4.4 above to confirm the expression of fibrosis-related proteins. The primary antibodies used were anti-Timp1 antibody (Cell Signaling, #63363), anti-α-SMA antibody (Abcam, ab5694), anti-Col1a1 antibody (Abcam, ab260043), anti-fibronectin antibody (Abcam, ab2413), and anti-GAPDH antibody (Cell Signaling, #2118). The secondary antibody used was anti-rabbit IgG HRP-binding antibody (Cell Signaling, 7074S).

[0120] As a result, as shown in Figures 26a and 26b, the expression of fibrosis-related proteins (α-SMA, COL1A1, Timp1, and fibronectin) was confirmed to be reduced in the BLM+hMSC-EV administration group compared to the bleomycin (BLM) administration group.

[0121] Example 6.2.4. Confirmation of soluble collagen concentration in lung tissue The lower and upper lobes of the lungs obtained from the experimental mice in Example 6.2.1 above were extracted and pulverized using a homogenizer. The soluble collagen concentration in the tissue was measured using the same method as in Example 6.1.3 above.

[0122] As a result, as shown in Figure 27, collagen concentrations in both the lower and upper lobes were found to be lower in the BLM+hMSC-EV administration group compared to the bleomycin (BLM) administration group.

[0123] Example 6.2.5. Confirmation of pathological morphology of lung tissue via hematoxylin and eosin staining. The left lobe of the lung obtained from the experimental mice in Example 6.2.1 above was extracted, and tissue sections were prepared using the same method as in Example 6.1.4 above, and stained with hematoxylin and eosin. At this time, the tissue sections were treated with Weigert's iron hematoxylin working solution, stained with eosin working solution for 30 seconds, and then washed in running water for 3 minutes. After that, dehydration was performed (ethanol 70% (2 min), ethanol 80% (2 min), ethanol 90% (2 min), ethanol 100% (2 min), xylene (3 min)), and then the sections were mounted and the pathological morphology of the lung tissue was examined under a microscope. In addition, the observation results were expressed as an Ashcroft score (Figure 28).

[0124] Example 6.2.6. Confirmation of collagen expression regions in lung tissue via Masson's trichrome staining. The collagen expression regions in the lung tissue obtained from the experimental mice in Example 6.2.1 were identified via Masson's trichrome staining using the same method as in Example 6.1.4.

[0125] As a result, as shown in Figure 29, the collagen region in lung tissue was significantly reduced in the BLM+hMSC-EV administration group compared to the bleomycin (BLM) administration group, and its level was confirmed to be equivalent to that of the normal group.

[0126] The results described above confirm that extracellular vesicles isolated from human mesenchymal stem cells have the effect of improving or treating pulmonary fibrosis.

Claims

1. An extracellular vesicle containing one overexpressed microRNA selected from the group consisting of hsa-miR-148a-3p, hsa-miR-100-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-let-7f-5p, and combinations thereof.

2. The extracellular vesicle according to claim 1, wherein hsa-miR-148a-3p is present in an amount of 1 to 20% of the total microRNA content of the extracellular vesicle.

3. The extracellular vesicle according to claim 1, wherein hsa-miR-100-5p is present in an amount of 1 to 20% of the total microRNA content of the extracellular vesicle.

4. The extracellular vesicle according to claim 1, comprising hsa-miR-143-3p in an amount of 0.1 to 10% of the total microRNA content of the extracellular vesicle.

5. The extracellular vesicle according to claim 1, comprising hsa-let-7a-5p in an amount of 0.1 to 10% of the total microRNA content of the extracellular vesicle.

6. The extracellular vesicle according to claim 1, comprising hsa-let-7f-5p in an amount of 0.1 to 10% of the total microRNA content of the extracellular vesicle.

7. The extracellular vesicle according to claim 1, wherein the microRNA exhibits fibrosis inhibitory activity.

8. hsa-miR-151a-3p, hsa-let-7b-5p, hsa-miR-21-5p, hsa-miR-10a-5p, hsa-miR-92a-3p, hsa-miR-486-5p, h sa-miR-99b-5p, hsa-let-7i-5p, hsa-miR-320a, hsa-miR-409-3p, hsa-miR-10b-5p, hsa-miR-127-3p, hsa-m iR-26a-5p, hsa-miR-221-3p, hsa-let-7e-5p, hsa-miR-493-5p, hsa-miR-25-3p, hsa-miR-574-3p, hsa-miR- 423-5p, hsa-miR-122-5p, hsa-miR-382-5p, hsa-miR-155-5p, hsa-miR-451a, hsa-miR-30a-3p, hsa-miR-28- 3p, hsa-let-7g-5p, hsa-miR-379-5p, hsa-miR-23a-3p, hsa-miR-485-5p, hsa-miR-30a-5p, hsa-miR-30d-5 p, hsa-miR-423-3p, hsa-miR-99a-5p, hsa-let-7c-5p, hsa-miR-92b-3p, hsa-miR-323a-3p, hsa-miR-574-5p The extracellular vesicle according to claim 1, further comprising one expressed microRNA selected from the group consisting of hsa-miR-197-3p, hsa-miR-432-5p, hsa-miR-140-3p, hsa-miR-370-3p, hsa-miR-196a-5p, hsa-miR-654-5p, hsa-miR-184, hsa-miR-181a-2-3p, and combinations thereof.

9. An extracellular vesicle isolated from human-derived stem cells, as described in claim 1.

10. The extracellular vesicle according to claim 9, wherein the stem cells are mesenchymal stem cells.

11. The extracellular vesicle according to claim 10, wherein the mesenchymal stem cells are derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, tooth, hair follicle cells, or placenta, or differentiated from induced pluripotent stem cells.

12. An extracellular vesicle according to claim 1, wherein the particle size is 10 nm to 1,000 nm.

13. A pharmaceutical composition for preventing or treating fibrosis, comprising the extracellular vesicle described in claim 1 as an active ingredient.

14. The pharmaceutical composition for preventing or treating fibrosis according to claim 13, wherein the fibrosis is pulmonary fibrosis.

15. A pharmaceutical composition for preventing or treating fibrosis according to claim 13, wherein the pulmonary fibrosis is one or more selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced lung injury, nonspecific interstitial pneumonia, acute interstitial pneumonia, idiopathic organizing pneumonia, interstitial lung disease with respiratory bronchiolitis, desquamative interstitial pneumonia, lymphocytic interstitial pneumonia, interstitial pulmonary fibrosis and diffuse pulmonary fibrosis, pulmonary edema, cystic fibrosis, and pulmonary fibrosis caused by metabolic disease.

16. Use of the extracellular vesicle according to claim 1 for the prevention or treatment of fibrosis.

17. A method for preventing or treating fibrosis, comprising the step of administering to extracellular vesicles as described in claim 1.

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